The first X-ray structure of a ternary adduct of human carbonic anhydrase II with the activator phenylalanine and the inhibitor azide is reported. Confirming what is suggested by spectroscopic data on the Co(II) derivative of HCA II, the phenylalanine molecule does not interact with zinc and it is positioned in the hydrophobic region of the active site cavity, similarly to what is observed for histamine, the first activator for which the X-ray crystallographic structure has been reported. The aromatic ring of phenylalanine is pointed towards the cavity opening while its amino group is involved in a strong hydrogen bond with a water molecule which, on the other hand, interacts with a zinc-bound azide anion. This result provides further support for the existence of an ‘activator binding site’ in the HCA II cavity which allows the binding of compounds able to provide alternative proton transfer pathways from the zinc-bound water molecule to the bulk solvent.